Treatment-matched minority variants signal delayed culture conversion in rifampicin-resistant tuberculosis

In a retrospective study of 229 patients with rifampicin-resistant or multidrug-resistant tuberculosis in Ningbo, China, treatment-matched minority resistance-associated variants detected at allele frequencies of 5% or more were associated with higher culture positivity six months after treatment began. The study found no clear overall association between aggregate minority-variant burden and microbiological response after adjustment for covariates. It also identified a higher positivity rate among patients who carried both a high burden of fixed resistance-associated variants and the treatment-matched minority variants, but the small exploratory groups and wide confidence interval preclude causal or clinical conclusions.

The researchers combined clinical, phenotypic, treatment, microbiological and whole-genome sequencing data from patients treated between 2021 and 2025. Routine interpretation of Mycobacterium tuberculosis resistance has focused mainly on consensus-level mutations, whereas minority variants are detected at lower allele frequencies and may represent resistant subpopulations under drug pressure. The investigators defined minority resistance-associated variants as low-frequency variants matching entries in the World Health Organization’s second version of its mutation catalogue and detected at allele frequencies of at least 3%.

A primary higher-frequency subset comprised variants found at allele frequencies of at least 5%. Per-drug analyses excluded patients whose target genes had a median sequencing depth below 200-fold. Associations with microbiological response were assessed using covariate-adjusted logistic regression, while higher-frequency and treatment-matched variants were examined in secondary analyses.

The clearest signal appeared among the seven patients with treatment-matched minority variants at allele frequencies of 5% or more. Four of these patients remained culture-positive at six months, compared with 40 of 198 non-carriers, corresponding to culture-positivity rates of 57.1% and 20.2%. The adjusted odds ratio for six-month culture positivity was 7.21, with a 95% confidence interval of 1.38 to 43.68; the reported p-value was 0.020 and the multiple-testing-adjusted q-value was 0.034.

Including this treatment-aware indicator produced only a modest increase in model discrimination, raising the area under the receiver operating characteristic curve from 0.675 to 0.695. Thus, the statistical association was not accompanied by a large improvement in the model’s ability to classify six-month microbiological status. The highest observed culture-positivity rate occurred among patients with both a high fixed resistance-associated-variant burden and treatment-matched minority variants at allele frequencies of 5% or more.

Fisher’s exact test yielded p = 0.003. These results show an association in a selected subgroup rather than evidence that minority variants caused delayed culture conversion. The retrospective design, small number of carriers, wide uncertainty around the adjusted effect estimate and potential influence of treatment and other clinical factors limit causal interpretation.

The authors characterized the findings as exploratory and said that external validation is required before clinical use; the study does not establish that routine detection of minority variants can predict individual outcomes or improve treatment decisions.

Subject of Research: Minority resistance variants and six-month culture conversion in rifampicin-resistant or multidrug-resistant tuberculosis.

Article Title: Resistance-gene minority variation and delayed microbiological response in rifampicin-resistant tuberculosis

Article References: Fang, Q., Li, X., Lu, Y., Zhang, D., Li, F., Gao, J., & Che, Y. (2026). Resistance-gene minority variation and delayed microbiological response in rifampicin-resistant tuberculosis. Frontiers in Cellular and Infection Microbiology, 16, 1930268. https://doi.org/10.3389/fcimb.2026.1930268

Image Credits: AI Generated

DOI: 10.3389/fcimb.2026.1930268

Keywords: rifampicin-resistant tuberculosis, multidrug-resistant tuberculosis, minority variants, whole-genome sequencing, culture conversion, allele frequency, antibiotic resistance, treatment response, Resistance-gene, minority, variation, delayed